Immunoglobulin (Ig) heavy chain locus (IgH) VH, D, and JH segments are clustered over megabase (Mb) regions of the mouse IgH locus. Their assembly via V(D)J recombination is regulated in multiple contexts, including ordered D to JH joining prior to VH to DJH joining, with the latter step regulated to promote utilization of proximal and distal VHs to promote diverse repertoires. The IGCR1 control region in VH to D inter-genic region mediates V(D)J recombination control via two CTCF-binding elements (CBEs) proposed to mediate regulatory chromatin loops. To initiate V(D)J recombination, the RAG1/2 endonuclease (RAG) cleaves V(D)J gene segments flanked by complementary recombination signal sequence (RSS) pairs and mediates their subsequent joining. RAG can directionally track across Mb chromosomal loops, which could allow it to locate proper RSS pairs for cleavage activities. RAG tracking was discovered with our new LAM-HTGTS technique, which is so sensitive that it reveals multitudes of low frequency RAG-initiated cleavage/joining events invisible to prior assays. We have adapted the LAM-HTGTS for V(D)J repertoire sequencing (?HTGTS-Rep-seq?). We propose four specific aims designed to build on these findings and new technologies to elucidate mechanisms that promote joining of IgH Vs, Ds, and Js across Mb distances to generate diverse antibody repertoires. We also propose to extend these studies to other mouse Ig loci and to human antigen receptor loci. Many proposed studies will employ G1-arrested, RAG-inducible v-Abl transformed pro-B cell lines as a test system, but key results will be confirmed or extended by studies of normal B lineage cells. Aim 1 proposes to elucidate the mechanism and functional consequences of RAG tracking by using a large c-Myc locus loop domain as a test system to obviate potential confounding effects of numerous RSSs and other regulatory elements in antigen receptor loci. Proposed studies will evaluate contribution of RAG tracking versus other mechanisms for capture of distant RSS pairs and elucidate factors that propel RAG tracking. Aim 2 proposes to elucidate mechanisms that regulate IgH D to JH recombination. In IgH, Ds are located upstream of JHs within an IGCR1- dependent domain that isolates them from upstream Vs. In this domain, D to JH recombination initiates from a RAG-bound recombination center (RC) comprising JHs and the most proximal D (DQ52). We will test the hypothesis that RAG is activated in the RC by pairing of DQ52 and JH RSSs, with convergent 12RSSs of other Ds then captured by other mechanisms, potentially including tracking. We also propose to define cis elements required for RC formation. Aim 3 proposes to elucidate mechanisms that bring distant VHs into V(D)J recombination domains, including unique aspects of IgH CBE organization across the IgH locus and potential roles of VH-associated CBEs. This aim also proposes to extend studies to the Ig? light chain locus. Aim 4 proposes to extend Aim 2 and 3 studies to human antigen receptor loci, which is important because V(D)J recombination control mechanisms of human Ig or TCR loci have not been studied directly.